Covering the bases: Population genomic structure of Lemna minor and the cryptic species L. japonica in Switzerland

Abstract Duckweeds, including the common duckweed Lemna minor, are increasingly used to test eco‐evolutionary theories. Yet, despite its popularity and near‐global distribution, the understanding of its population structure (and genetic variation therein) is still limited. It is essential that this is resolved, because of the impact genetic diversity has on experimental responses and scientific understanding. Through whole‐genome sequencing, we assessed the genetic diversity and population genomic structure of 23 natural Lemna spp. populations from their natural range in Switzerland. We used two distinct analytical approaches, a reference‐free kmer approach and the classical reference‐based one. Two genetic clusters were identified across the described species distribution of L. minor, surprisingly corresponding to species‐level divisions. The first cluster contained the targeted L. minor individuals and the second contained individuals from a cryptic species: Lemna japonica. Within the L. minor cluster, we identified a well‐defined population structure with little intra‐population genetic diversity (i.e., within ponds) but high inter‐population diversity (i.e., between ponds). In L. japonica, the population structure was significantly weaker and genetic variation between a subset of populations was as low as within populations. This study revealed that L. japonica is more widespread than previously thought. Our findings signify that thorough genotype‐to‐phenotype analyses are needed in duckweed experimental ecology and evolution.

species retain genetic diversity, that is, plant populations are often multiclonal and clones are highly population-specific (Ellstrand & Roose, 1987).But how this impacts eco-evolutionary findings remains largely unclear, as molecular markers sensitive enough to characterize finer-scale variation within clonal species and populations have only recently become accessible (Anisimova, 2019;Ehrlich, 1988).Thus, practical examples of intraspecific genetic diversity impacts lag behind our theoretical understanding of its importance.Studies to fill this gap are crucial, especially when they provide a foundation to elucidate the adaptive significance of genetic diversity in changing environments.
Researchers increasingly rely on model systems like duckweed to gain insight into species responses to environmental change.
Duckweeds (Lemnoideae) are a group of flowering aquatic plants in the Araceae family that float on the surface of still or slowmoving freshwater bodies (Landolt, 1986).They are monocotyledonous, represented by 37 species (Sree et al., 2016), including an interspecific hybrid, Lemna japonica, with L. turionifera and L. minor as parents (Braglia et al., 2021).Lemna spp.often occur at high densities in ponds, streams, and ditches in a wide range of environmental conditions (Landolt, 1975), including polluted waters.They disperse through wind, water, birds, and, increasingly, humans' activities.Duckweeds are of high interest for applied conservation in wastewater treatment, so-called phytoremediation (Sekomo et al., 2012).They also hold commercial potential as a protein source for food products (e.g., as a soy substitute, Sree et al., 2016) and as biofuel.Lemna spp.have the potential to be used for revealing plant defense strategies, genome maintenance, and other complex plant biochemical processes (Acosta et al., 2021).Duckweeds, such as L. minor, also have a long history of use as model organisms in fundamental research and ecological experiments due to their small size, clonal propagation, and fast clonal generation time (as little as three days; Clatworthy & Harper, 1962;Hart et al., 2019).Recently they have increased in popularity as model system for experimental evolution, as well as evolution of species interactions (Hart et al., 2019;Laird & Barks, 2018;Lanthemann & van Moorsel, 2022;O'Brien, Laurich, et al., 2020;O'Brien, Yu, et al., 2020;Tan et al., 2021).
Lemna minor reproduces vegetatively by budding, with occasional sexual reproduction through flowering described in wild populations (Hicks, 1932;Landolt, 1986).The actual frequency of flowering is unclear and likely underestimated because the very small flower is hardly visible by eye (Ziegler et al., 2023).
Pollination of flowers occurs through wind, water, or small animals or by direct flower contact.It is still unknown whether L. minor is self-compatible, but self-pollination has been shown to result in sterility in the closely related species L. gibba (Fu et al., 2017).
Despite the dominance of asexual reproduction, L. minor may thus maintain relatively high levels of intraspecific genetic diversity for a predominately clonal species (Ho, 2018;Vasseur et al., 1993;but see Jordan et al., 1996).Importantly, there is evidence suggesting standing intraspecific genetic variation impacts fitness within populations (Jewell & Bell, 2023).In allozyme loci, intraspecific genetic diversity of duckweed is low within populations (i.e., individual water bodies like ponds) but between populations can be highly differentiated (Cole & Voskuil, 1996).Populations in close geographic proximity can harbor very different genotypes or clones (Hart et al., 2019;Ho, 2018;Tan et al., 2021;Vasseur et al., 1993) and may even be locally adapted to different environmental conditions (e.g., copper pollution, Roubeau Dumont et al., 2019).Recent studies have begun to leverage the substantial phenotypic variation in L. minor (Hart et al., 2019;Roubeau Dumont et al., 2019;Tan et al., 2021) but the intraspecific genetic variation, that is, presence of different alleles or the population genetic structure which may drive such phenotypic variation, is still not fully understood due to a lack of high-resolution population genetic studies on the species.Importantly, it is widely assumed that while L. minor populations are genetically differentiated between sites they are not differentiated within site (Cole & Voskuil, 1996).However, recent evidence found that multiple clones can co-occur in a single pond (Bog et al., 2022).Discrepancies in previous findings may be due to the fact that many genetic studies have examined a limited portion of the species' range, and importantly, used low-resolution genetic markers, which are not intercomparable, such as allozyme loci (Cole & Voskuil, 1996;Vasseur et al., 1993), ISSR markers (El-Kholy et al., 2015), Amplified Fragment Length Polymorphisms (Bog et al., 2022), or microsatellite loci (Hart et al., 2019;Kerstetter et al., 2023;Usui & Angert, 2024).Only recently genomic tools have been applied to this model system, a genotyping-by-sequencing study identified three genetically distinct populations of L. minor within a 160 km transect in Canada (Senevirathna et al., 2023).
Whole-genome sequencing (WGS) is a very powerful tool, capable of revealing complex or fine population structures in species with varying reproductive systems that include geographic clonality (Stauber et al., 2021) or weak population structure (e.g., Kersten et al., 2021).In this study, we used WGS to quantify the intra-specific genetic diversity of L. minor present in the species' described distribution in Switzerland to understand the structure and patterns in its genetic diversity and gain insights into how this could impact eco-evolutionary research.This is particularly pertinent because the mutation rate for the related duckweed species Spirodela polyrhiza is low (Ho et al., 2019;Wang et al., 2014).
Though mutation rates remain unknown for L. minor, they are thought to be equally low and thus evolution in L. minor during experiments will arise from standing genetic variation.Knowing whether this standing genetic variation can be manipulated by using different locally sourced ecotypes (e.g., as done in Hart et al., 2019;van Moorsel, 2022) or by collecting a large number of individuals from one site, is critical information for duckweed researchers.This led us to specifically test the hypothesis that intraspecific genetic diversity within a site is low or virtually absent, but large between sites.
Many species of the duckweed family are morphologically so similar that they are virtually impossible to distinguish with morphology alone (De Lange & Pieterse, 1973;Landolt, 1975).
Consequently, there are also doubts about whether the species has been correctly identified in past experimental studies.A screening of 100 clones from the Landolt duckweed collection found that several populations of L. minor had been misidentified based on morphological criteria (Braglia et al., 2021).A study in Canada has revealed the presence of a cryptic species (L.turionifera) using DNA barcoding with species-specific primers (Senevirathna et al., 2021).Lemna minor intraspecific diversity may have thus previously been overestimated due to the inclusion of cryptic species (Jordan et al., 1996).Within Switzerland, range maps indicate a prevalence of L. minor in Switzerland but very few records of closely related Lemna species (e.g., morphologically similar Lemna japonica, L. minuta, L. gibba, and L. turionifera).Difficulties surrounding correct species identification are likely leading to incorrect distribution maps, and there is a need for large-scale genetic screening of wild Lemna populations (Volkova et al., 2023).In line with this, our second research question was to find out whether there are cryptic species present in Switzerland.

| Sample collection
Lemna minor samples were collected opportunistically from 23 sampling sites (hereafter "wild populations") across a 200 km range within Switzerland and one location in France during July to September of 2021 and 2022 (Figure 1, Table 1).Sites ranged from 5 km to >100 km apart, with some separated by mountain ranges (Figure 1).Within sites, several microsites (samples) were collected (see also Figure S1).Given that all individuals of a sample grew close to each other, we expected that each sample contained only single clones.While it is possible that with our sampling approach we pooled different genotypes, we would expect differences in the allelic rations in such genotype mixtures and assumed this would be highly visible.However, except for one sample (GR6), allelic ratios were highly similar between samples of a given species and were also similar to the pattern observed in the strains that were cultivated and kept as single clone (see Results).Thus, it seems unlikely that we pooled more than one clone per sample (expect for GR6).
For each sample, 80 plant individuals were flash-frozen in liquid nitrogen.In addition to the wild populations, several Lemna clones from the Landolt duckweed collection were sampled and sequenced (also 80 individuals per sample).Each strain represented true clones with a recorded history in lab culture and scientific use.To act as reference outgroups, we included the following strains from candidate cryptic species: Lemna minuta (strain 9700 collected in Rotterdam, the Netherlands), L. japonica (strain 9983 collected in Meilen, Switzerland), and L. turionifera (strain 9478 collected in Raciborz, Poland).We aimed to include L. gibba; however, the strain we used (9965 collected in St. Gallen, Switzerland) was re-classified during our study as L. japonica (Braglia et al., 2021) based on Tubulin Based Polymorphisms (TBP).Furthermore, as described by Braglia et al. (2021) two of the used L. minor strains were reclassified as L. japonica (strains 9969 and 9978).All samples of the wild L. minor populations and the duckweed stock populations were kept at −80°C for downstream applications.In total, 87 samples (each based on 80 pooled plant individuals) that yielded high-quality data for genomics were sequenced (see Table 1).

| DNA extraction
For DNA extraction, 30 mg of the frozen tissue consisting of several fronds was weighed into 1.5 mL tubes (Eppendorf, Hamburg, Germany) with two 3 mm metal beads and ground into a fine powder with a TissueLyser II (Qiagen, Germany) at 30 Hz for 2 min.The extraction was performed using the Norgen Plant and Fungi genomic kit (Norgen Biotek, Thorold, ON, Canada) with minor modifications.Specifically, 750 μL of lysis buffer supplemented with 1 μL RNAse A (DNase-free, 100,000 units/mL in 50% glycerol) and 3 μL Proteinase K (>600 u/mL-20 mg/mL in 10 mM Tris-HCl pH 7.5, containing calcium acetate and 50% v/v glycerol) was added to each tube and vortexed vigorously.Then, 150 μL of Binding Buffer I was added to each tube, vortexed, and incubated for 5 min on ice.The rest of the extraction procedure was conducted according to the manufacturer's protocol.
Finally, the purified DNA was eluted in 100 μL of elution buffer and stored at −20°C.Quantification and quality control of the purified DNA was performed by Qubit dsDNA HS Assay Kits (Thermo Fisher Scientific) and gel electrophoresis.Library preparation and sequencing was conducted by Novogene (UK) using Illumina NovaSeq PE150 paired-end sequencing (Novogene, Cambridge, UK).

| K-mer-based approach
To process samples for population genetic analysis in a referencefree manner, we used a recently described pipeline (Voichek & Weigel, 2020, github.com/ voich ek/ kmers GWAS).Analyses were conducted using the default k-mer size of 31.The kinship matrix generated with the pipeline was converted to a distance matrix (1-kinship) to enable comparability across analyses.It should be noted that we did not intend to provide a full comparison between k-mer and alignment-based pipelines but to use a reference-free approach to assist with species misassignments.

| Reference-based approach
We used the reference genomes of either L. minor strain 7210 or the diploid L. japonica strain 9421 (Ernst et al., 2023).The L. minor assembly includes 21 chromosomes (362.1 million bp, all sizes haploid), 30 unassigned scaffolds (0.9 mio bp), and the plastid genomes (0.4 mio bp).The L. japonica assembly includes 21 chromosomes from the L. minor parent (347.0mio bp), 21 chromosomes from the L. turionifera parent (420.3mio bp), 85 unassigned scaffolds (3.2 mio bp), and the plastid genomes (0.4 mio bp).Reads were aligned using bowtie2 (version 2.4.5, with a minimal mapping quality of 10, Langmead & Salzberg, 2012).Runs of samples with more than one sequencing run were merged with samtools (version 1.18, Danecek et al., 2021).SNPs were identified with bcftools (version 1.18, Danecek et al., 2021) and filtered for a minimal quality of 20.SNPs were further filtered for a coverage between 10 and 1000 within at least 80% of all samples.Pairwise genetic distances were calculated as the fraction of alleles that differed between two individuals, while ignoring SNPs with pairwise missing data.After identification of the L. minor and L. japonica subgroups (see below), the subgroups were divided and treated separately.Each cluster was re-analyzed as described above using the species-specific genome.

| Non-plant DNA
To assess whether the samples contained sequences from other organisms, we scanned all reads with the metagenome classifier tool CLARK (version 1.2.6.1,Ounit et al., 2015).We used the family-level databases of fungi, bacteria, and viruses (set_targets.shoptions: bacteria viruses fungi --family) and a k-mer size of 31 (classify_metagenome.sh options -k 31 -m 2 --ldm --light).Abundances were obtained with default settings (estimate_abundance.sh).The fraction of reads that were assigned during the process was extracted from the output of classify_metagenome.sh.We filtered for families with at least 10 sequence counts in 10 samples and used DESeq2 (version 1.24.0,Love et al., 2014) to obtain log2 (x + 1) transformed normalized counts that were used to calculate pairwise Manhattan distances between the samples.Differential abundance of families between L. minor and L. japonica individuals was done as described previously (Schmid et al., 2019), and families with an FDR < 0.05 and a log2 fold-change of at least 2 were considered to be differentially abundant.
F I G U R E 1 Sampling sites.Cyan for sites with only Lemna minor individuals, yellow for sites with only L. japonica individuals, magenta for sites with individuals from both species.Species assignment based on the kmer-approach.

| SNP-wise test for association with genetic clusters ("assignment markers")
To test for associations of a genetic variant with a group of samples (i.e., the two genetic clusters which corresponded to the two species L. minor and L. japonica), we ran an ANOVA for each individual SNP using R (version 4.2.3, i.e., we ran a GWAS using a simple GLM).
The genetic variants in case of bi-allelic SNPs have three possible levels corresponding to "homozygous reference," "heterozygous," and "homozygous alternative": 0/0, 0/1, and 1/1.Besides this threelevel factor, we also used two two-level factors for comparisons of one homozygous state to the other two states (factor A: 0/0 vs. 0/1 and 1/1; factor B: 1/1 vs. 0/1 and 0/0).We then only considered the factor with the best fit.Factorial data was tested with ANOVA (glm (formula, family = quasibinomial("logit")), test = "F") and the effect size was calculated as the percent of deviance (%-DEV) explained by the variant.p-values were adjusted for multiple testing to reflect false discovery rates (FDRs).Associations with an FDR below 0.05 and %-DEV of at least 80% were considered significant.We chose the high %-DEV cut-off to only include cases that were clearly and TA B L E 1 Sampled sites, collection dates, and number of samples included in the data analyses.consistently different between the two species.Given the relatively large sample size, only filtering by FDR would also yield SNPs that are specific to for example one population and not the "entire" species.

| Differences in variation among populations
We tested whether the intra-population variation differed among populations using the function betadisper() from the R-package vegan (version 2.5.7,Dixon, 2003).The function was also used to extract the distances to the overall species' centroid by treating the entire species as one population (used for plotting).

| Isolation by distance
Isolation by distance, which involved the correlation between genetic distance matrices and the correlation of genetic distances with physical distances, was tested with Mantel tests using 9999 permutations with the R-package vegan (version 2.5.7,Dixon, 2003).
Correlations between genetic and physical distances were also tested with an ANOVA (anova(lm())).In both cases, physical distances in meters were log10 transformed.

| Visualizations
Heatmaps were generated using the distance matrices and the function gl.plot.heatmap()from the R-package "dartR" (version 1.0.2,Gruber et al., 2018).The genetic composition of the individual samples in Figure 3a was inferred using fastSTRUCTURE (version 1.0, Raj et al., 2014) as described in the online manual (rajanil.github.io/fastStructure).The number of populations was set to K = 8 (L.minor) and K = 4 (L.japonica) because this was the number of expected populations based on the distance visualizations (Figure 3). Figure S4B was done using the function heatmap.2 from the R-package gplots (version 3.1.1,Warnes et al., 2024).All other plots were done in R using base graphics.

| Three different approaches recovered two species
The kmer approach identified 1,357,142,099 variants in the kmer table.
Alignment to the Lemna minor reference genome (strain 7210) and SNP calling with bcftools (Danecek et al., 2021) 2a).To test whether these two clusters could be attributed to two species, we used three additional approaches: (1) alignment rate comparison, (2) identification of clusterspecific SNPs, and (3) comparison of alternative allele depths using also publicly available data (Ernst et al., 2023).

| Differences in alignment rates
Alignment rates for the L. japonica reference genome ranged from 50% to 90%, except for the L. minuta sample which had an alignment rate of 2.9%.Rates for the L. minor genome were generally lower for all samples, but there was a marked difference among samples (Table S1, Figure S2).
While the alignment rates of the L. minor samples were on average only 10.2% lower (range: 5.6-13.9%),alignment rates of L. japonica samples were about 26.1% lower (range: 16.3-32.4%).As a comparison, the alignment rate of the L. turionifera sample, whose genome is only represented in the L. japonica reference, was 66.1% lower.

| Species assignment markers
We tested all SNPs that passed the filter for significant association with either of the clusters (i.e.species) using generalized linear models (GLMs) followed by multiple testing corrections and stringent filtering (FDR < 0.05 and %-deviance explained >80%).While using the L. minor reference, 5.95% of all SNPs were highly differentiated between the two clusters.In contrast, with the L. japonica reference only 0.13% of all SNPs showed significant differentiation (Figure 2b).Meeting with theoretical expectations for allele frequencies between species, most differentiated markers were homozygous fixed differences (99.76% L. minor reference, and 82.30% L. japonica reference).The difference in the fraction of significant SNPs was probably due to the fact that parts of the genome from L. japonica (i.e., the copy from the parent that is not L. minor, hence L. turionifera) wrongly aligned to the L. minor reference sequence and that this was corrected when using the L. japonica reference genome.The limited difference between the two clusters with the L. japonica reference genome further suggested that the copy of the L. minor parent in the L. japonica individuals was similar to the genome of the L. minor individuals.

| Alternative allele ratio suggests that L. japonica is triploid
To verify the difference between the two clusters and assess the genetic composition of the L. japonica individuals, we extracted the "alternative allele ratio" at each SNP using the L. minor reference genome data (Figure 2c).For example, one SNP might be covered with 30 reads that suggest that the base is "A" like in the reference genome, whereas another 30 reads suggest an alternative base "T." Hence, the ratio would be 0.5 and indicate that the individual is heterozygous at this position.However, if there were only 15 reads suggesting the base "T," the ratio would be 1:2 and indicate that the individual might be triploid with one copy being different (or hexaploid with two copies being different).Doing this for all SNPs, we indeed found an enrichment of the alternative allele ratio of 50% in all L. minor individuals and an enrichment of an alternative allele ratio around 33% in all L. japonica individuals (Figure 2c).To verify this approach, we added data from the previously published Lemna reference genomes (Ernst et al., 2023; Figure S3).Among them The correlation was significant in both species (both p Mantel < .01 and both p ANOVA < 10 −10 ) but much higher in L. minor (Pearson correlation of .83)compared to L. japonica (Pearson correlation of .24).
Considering that L. japonica was a hybrid with the genomes of both parents available in the assembly, we could compare the variation within these two "sub-genomes."We, therefore, split the SNPs according to the sub-genome of origin, re-calculated the pairwise distances, and compared the distances to each other.If the two were markedly different from each other, it might indicate that the data contained samples with different parents.Due to differences in coverage (Figure S3), the number of SNPs was significantly different: 516,007 and 4081 in the L. minor (M) and the L. turionifera (T) sub-genome, respectively.Nonetheless, pairwise distances were very similar to each other (Pearson correlation of .95,p Mantel = .0001with 9999 permutations).It is therefore possible that the L. japonica samples have a common hybrid ancestor and that the differences we observed were gained after the original hybridization.However, it is also still possible that there were multiple hybridization events but that the dissimilarity between the parents was similar.
Similar to comparing the variation between the two sub-genomes, we further extracted SNPs in the mitochondrial and chloroplast genomes, and re-calculated the pairwise distances using these subsets.
Distances based on the plastid genomes were similar to each other (Table 2).When distances based on plastids were compared to distances based on the M and T sub-genomes, correlations between the mitochondrial and the nuclear genomes were higher than correlations between the chloroplast and the nuclear genomes.However, in both cases, correlations to the M sub-genome were clearly higher than those to the T sub-genome (Table 2).This suggests that the female parent that contributed to the plastid genomes was likely L. minor.This is further supported by visualizing the similarity in the plastid genomes using all samples, including publicly available data (Figure S3D).While all L. minor and L. japonica samples are similar, the two L. turionifera and the L. minuta samples are very distinct.This is especially interesting considering that the publicly available L. japonica samples have different subgenome compositions (MT, MMT, and MTT).
In summary, we found clear population structures that also reflected the physical distance between the populations, especially in L. minor.In L. japonica, the structure was less pronounced, and several populations were similar enough to be grouped into one large group exhibiting "within-population-level" genetic differences even though they were physically well separated from each other ("various populations" in Figure 3a).

| Non-plant eDNA associated with collected samples
On average, about 6.19% of all reads were classified as bacterial, viral, or fungal and the two species were alike (Figure S4A).In total, 555 different fungi/bacteria/virus families passed the filters, resulting in a table with varying read counts per sample and fungal/bacterial/viral family.Pairwise Pearson correlation coefficients between samples using these data were between .67 and .99.Samples were not grouped by eDNA according to populations, but to some extent by species (Figure S4B).Likewise, correlations between pairwise distances from the eDNA data to the three approaches above were limited (but p Mantel < .01):with the L. minor genome: 0.17, with the L.
japonica genome: 0.20, and with kmers: 0.09.Thus, the eDNA in the samples did not reflect the population structure observed with the plant data.Averaged L. minor and L. japonica samples were highly similar to each other (correlation of .98, Figure S4C).Out of the 555 families, five showed differential abundance in L. japonica compared to L. minor.All five families were more abundant in L. japonica samples: Desulfuromonadaceae, Fastidiosibacteraceae, Paludibacteraceae, Prolixibacteraceae, and Marseilleviridae.While little is known about these families, there are differences for some families and the distances in eDNA did not simply reflect those obtained from the plant data.This suggests that a proportion of reads originated from organisms other than the studied plants.While this may be used to study the microorganisms associated with the plants, it also indicates that the kmer approach should be used carefully as it does not inherently filter against sequences coming from non-target organisms.An option might be to filter reads matching to eDNA before running the kmer analysis.

| DISCUSS ION
Within this study we used sampling of natural sites of Lemma minor in Switzerland and France, coupled with whole genome sequencing, to characterize population structure and standing genetic variation of this common model system.We found expected patterns of low within site variation relative to between site differentiations in the species.Perhaps, most strikingly, we found much of the described distribution of L. minor actually contained L. japonica, with the two species also showing different patterns of differentiation.
TA B L E 2 Pairwise distances using only SNPs in specific sub-genomes of the Lemna japonica assembly.(Bog et al., 2022;Cole & Voskuil, 1996;Hart et al., 2019).However, more genetic variation was found here relative to the comparable geographical range reported in Senevirathna et al. (2023).These results indicate that when including populations from multiple sites in an experimental setting, chances are high that these combined populations contain multiple clonal genotypes.This will result in a baseline population with some standing genetic variation on which selection can act, the exact level of which will depend on the number of sites mixed and the distance between them.However, when including only individuals from one site (i.e., a single population), there is a high chance that the individuals are genetically very similar to each other, if not clonal.This difference in baseline will likely impact the obtained results (van Moorsel, 2022).Furthermore, knowledge of the standing genetic variation is needed for a mechanistic understanding of adaptive processes in experimental evolutionary studies (Barrett & Schluter, 2008;Lemmen et al., 2022;Usui & Angert, 2024).

| Presence of cryptic species revealed
In this study, we identified that a large number of sites were L. japonica.Indeed, we showed the presence of both L. minor and L. japonica is widespread in Switzerland.This was surprising, as current species range maps of L. minor, such as for example provided by Info Flora (https:// www.infof lora.ch/ de/ flora/ lemna -minor.html), indicate a broad distribution of L. minor across Switzerland but no records of L. japonica.Correctly attributing species within the Lemna family is challenging (Volkova et al., 2023).However, our results are supported by recent evidence; the presence of L. japonica in Switzerland was recently inferred based on the re-analysis of strains from the Landolt duckweed collection that showed a number of species misassignments (Braglia et al., 2021).However, the large geographical extent of L. japonica's distribution was unknown prior to our study.
The substantial geographical range shown here indicates duckweed research should molecularly confirm the focal species.This ensures accurate conclusions are drawn and experimental findings are attributed to the correct species.Perhaps also due to erroneous species attribution, little is known about how L. minor and L. japonica vary in traits relevant for practical purposes.Recent evidence suggests that the two species may vary in functional traits, such as relative growth rate or thermal performance breath (Usui & Angert, 2024).
Generally, closely related species of duckweed are known to exhibit strong differences in phenotypic traits, such as for example tolerance to pollutants (Lahive et al., 2011), and it remains to be tested whether this also holds true for L. minor and L. japonica.

| Population genomic structure of L. japonica
In contrast to L. minor, the population genomic structure of L. japonica is less clearly defined.Many sites were highly similar to each other (on the level of intrapopulation variation) and thus potentially clonal.However, this was spatially variable with some groups of sites showing levels of differentiation like the patterns seen in L. minor.This is in line with the hypothesis that hybridization between L. minor and L. turionifera has occurred multiple times, leading to independent lineages of L. japonica with different ploidy or chromosome rearrangements (Braglia et al., 2021;Ernst et al., 2023).However, it is unlikely these have arisen recently in situ in Switzerland because the sampling campaign did not recover any of the parent species L. turionifera.Furthermore, L. turionifera is thought to be currently and historically only present in three sites across all of Switzerland (https:// www.infof lora.ch/ de/ flora/ lemna -turio nifera.html).Taken together, this suggests that the sampled L. japonica hybrids arose outside of Switzerland and were dispersed by metazoan activity.
We compared our L. japonica samples to multiple hybrid L. ja-  (Stauber et al., 2021).
Our findings suggest that all L. japonica samples were triploid, which may explain the weaker population structure in L. japonica as compared to L. minor.Triploidy is often associated with infertility and vegetative propagation (Ramsey & Schemske, 1998), indicating that L. japonica is more likely to be clonal than its diploid sister species.
Polyploidization is assumed to be one of the major mechanisms of plant speciation and evolution (Herben et al., 2017).The relative importance may be greatest for plant species which reproduce mostly vegetatively, and thus experience less genome mixing via sexual reproduction.L. minor and L. japonica could thus present an interesting comparative system to study the evolution of sex.
It remains to be tested whether this L. japonica population genomic structure is ubiquitous to the species or specific to the unique geographical structure or colonization history of Switzerland.Recent interest in this species for technical applications (Liang et al., 2023) certainly warrants a deeper investigation.It might also be worth to consider studying DNA methylation as this can resolve intraspecific phylogenies with a high temporal resolution (Yao et al., 2023).

| CON CLUS IONS
We used whole-genome sequencing to elucidate the population genomic structure of L. minor in Switzerland.Our analyses revealed the presence of L. japonica in Switzerland.Consistent with previous studies, we conclude that the morphological analysis alone may not be sufficient for accurate species attribution within Lemna spp. in the field.Future studies using Lemnaceae as a model system in experimental population genetics (Acosta et al., 2021), community ecology (Laird & Barks, 2018), and eco-evolutionary dynamics (Hart et al., 2019) or applied research should ensure the accurate identification of L. minor.Our study offers valuable genomic resources for researchers working on Lemna species.Whole-genome sequencing has become a cost-effective and powerful tool to describe duckweed population structure and should be used in future efforts to map species and genotype presence of Lemna species.
were three different L. japonica hybrids with different mixtures of the parental genomes M (L.minor) and T (L.turionifera): (1) a diploid M/T, (2) a triploid M/M/T, and (3) a triploid M/T/T hybrid.These three hybrids matched the order of the expected alternative allele ratios with the lowest in M/M/T, intermediary in M/T, and highest in M/T/T.While the expected ratios would be in theory 33, 50, and 66%, the observed ratios were slightly lower (see FigureS3).It is unclear why this was the case.Nonetheless, all L. japonica in our study were closest to the triploid M/M/T hybrid, indicating that L. japonica in Switzerland also comprises triploid M/M/T hybrids.Additional support for this hypothesis came from the average coverage of the two sub-genomes, L. minor and L. turionifera, in the L. japonica reference genome.While the L. minor samples had practically zero coverage on the L. turionifera sub-genome, the L. japonica samples exhibited approximately a 2:1 ratio in genome coverage between the L. minor and the L. turionifera sub-genomes (FigureS3C).Taken together, our analyses suggest the widespread presence of a triploid L. japonica hybrid with two copies of the L. minor F I G U R E 2 Analysis with all samples.(a) Kinship matrices based on kmers, alignment with Lemna minor reference genome (Lm7210), and alignment with L. japonica reference genome (Lj9410).Species were assigned based on the kmer-approach and match the approach with L. minor as the reference genome.(b) Test for genetic differentiation between L. minor and L. japonica on the level of individual SNPs.Significance level was set to FDR <0.05 and %-deviance explained by the species >80%.Most differences were homozygous versus heterozygous differences: 99.73% and 80.69% in case of the L. minor and the L. japonica reference genome, respectively.(c) Allele frequencies in L. minor versus L. japonica individuals using the L. minor reference genome.Lemna minor individuals have an enrichment of non-homozygous frequencies at 50%.In contrast, L. japonica individuals have an enrichment of non-homozygous frequencies of around 33% (see also FigureS3with data from the publicly available reference genomes).
figuration as the isolate from Denmark (L.japonica 8627).The other hybrids are from Africa and North America and have different subgenome dosages.Broader geographical comparisons of L. japonica individuals with same ploidy level and configuration (M/M/T) are now needed to confirm if multiple hybridization events lead to different L. japonica lineages or if all the L. japonica individuals of the M/M/T configuration have a single common ancestor.This is relevant as different clonal lineages may behave differently depending on their ancestry as has previously been observed in fungal clones of C. parasitica(Stauber et al., 2021).

Higher between-than within population genetic variation in L. minor
Lemna minor generally displayed very low within population genetic diversity.It is noteworthy that the genetic distances reflect the fraction of alleles that differ between individuals and are calculated only on positions where at least one individual has an alternative allele.